Identification of Sulfamoylbenzamide derivatives as selective ...

Identification of Sulfamoylbenzamide derivatives as selective
Cathepsin D inhibitors
Waseem Ahmed *1,2 , Ishtiaq Ahmad Khan 1 , Muhammad Nadeem Arshad 3 ,
Waseeq Ahmad Siddiqui 3 , Muhammad Abdul Haleem 4 and Muhammad Kamran Azim 1
1 Dr. Panjwani Center for Molecular Medicine and Drug Research, ICCBS, University of Karachi, Karachi, Pakistan
2 Department of Biochemistry, Fed. Urdu Univ. of Arts Sci. & Tech., Gulshan-e-Iqbal Campus, Karachi, Pakistan
3 GC University, Ketchery Road, Lahore, Pakistan
4 Department of Biomedical Engineering, Sir Syed University of Technology, Karachi, Pakistan
Abstract: Aspartic proteases play very important role in post translational processing of proteins and several of them are
essential for organism’s viability. Here we present the enzyme inhibition activities of different Sulfamoylbenzamide
derivatives against two aspartic proteases cathepsin D and plasmepsin II. Cathepsin D is an aspartic protease that
degrades proteins at acidic pH in the lysosomes, or extracellular matrix. It is overexpressed by epithelial breast cancer
cells and hence hyper-secreted. On the other hand plasmepsin II is an essential enzyme of Plasmodium falciperum.
Cathepsin D and Plasmepsin II are pivotal drug targets for treatment of breast cancer and malaria respectively. Virtual
screening of Sulfamoylbenzamide compounds followed by enzyme inhibition assays revealed these compounds as
selective Cathepsin D inhibitors while inactive against Plasmepsin-II. IC50 values of five Sulfamoylbenzamide
compounds tested are in range of 1.25-2.0 μM. N-(3-chlorophenyl)-2-sulfamoylbenzamide is identified as the most
potent of all tested Sulfamoylbenzamide compounds with IC50 1.25 μM. It was also noted that the docking score
of theses compounds was better in case of Cathepsin D as compared to Plasmepsin-II. Docking score ranges from
-29.9±1.16 to -35.1±0.13 in case of Cathepsin D, while from -24.0±0.10 to -29.5±0.10 in case of Plasmepsin-II.
Keywords: Aspartic proteases, Cathepsin D, Plasmepsin-II, enzyme inhibition.
INTRODUCTION
Proteases control several key functions of cells such as
extravasation, invasion, motility, proliferation and
metastasis (Smith and Marshall 2010; Roy et al., 2009;
Palermo and Joyce 2008). Among these, cathepsin
proteases play main regulatory role as the factor-binding
proteins and growth factors and their receptor activities
have a major biological function in development of
human colorectal cancer (Sakkiah et al., 2012., Masson et
al., 2010; Rawlings and Barrett, 1993). Human cathepsin
D is an aspartic protease which functions at acidic pH
extracellularly in the matrix or lysosomes. It have been
demonstrated by several studies that Cathepsin D affects a
range of steps in tumor metastasis and progression. It
stimulates cancer cell growth by tumor angiogenesis
stimulating fibroblast growth factors (Benes et al., 2008;
Masson et al., 2010). Endosomes and lysosomes retain
Cathepsin D in normal cells but in breast cancer cells it is
over expressed and hyper-secreted by epithelial breast
cells (Liaudet-Coopman et al., 2006, Azim et al., 2008;
Gasparini 1998). Consequently degradative capacities of
these cells are changed along with accumulation of
Cathepsin D in the tumor microenvironment. Lysosomal
Cathepsin D is secreted into the cytosol during apoptosis,
where it may interact with and/or cleave nuclear anti-
*Corresponding author: e-mail: Hwast_9@hotmail.com
Pak. J. Pharm. Sci., Vol.26, No.4, July 2013, pp.687-690
apoptotic or pro-apoptotic proteins. During the resolution
of inflammatory responses, neutrophils rapidly undergo
apoptosis. A direct and fast activation of caspase-8 by
cathepsin D has been shown to be crucial in the initial
steps of neutrophil apoptosis (Conus et al., 2012).
The plasmepsin family of Plasmodium aspartic proteases
is involved in haemoglobin degradation during the intraerythrocyte
phase of malarial infection (Ersmark et al.,
2006). Plasmepsins have been considered as promising
target for new anti-malarial drugs (Ahmad et al., 2010).
The bilobal structure of Cathepsin D and plasmepsin II
proteases contain predominantly b-sheets with small ahelical
segments. The active site is located at the interface
between the two domains encloses two invariant
aspartates. Despite the structural similarities among the
members of aspartic proteases they have for less sequence
similarities. For example, Cathepsin D exhibits about
35% sequence homology to Plasmepsins (Schechter and
Berger 1967; Silva et al., 1996).
Benzenesulfonamide derivatives have a great potential for
medicinal applications (Ashraf et al., 2012; Siddiqui et
al., 2012, Siddiqui et al., 2010, Siddiqui et al., 2008,
Siddiqui et al., 2007). The sulfamoylbenzamide
compounds are agonists and modulating ligands of
cannabinoid receptors. These compounds are considered
687

Identification of Sulfamoylbenzamide derivatives as selective Cathepsin D inhibitors
Table 1: Docking scores and inhibition data of selected Sulfamoylbenzamide compounds
W-5
688
Compd. ID Structure
O
N
H
OH
O
O
S
O
NH2 3-(2-sulfamoylbenzamido)benzoic acid
H
N
N
W-7 S
O
O
N-benzyl-1,2-benzothiazol-3-amine 1,1-dioxide
O
W-24
W-28
W-30
N
H
S NH
Cl
2
O
O
N-(3-chlorophenyl)-2-sulfamoylbenzamide
Docking in CD
(FlexX score)
useful for gastrointestinal disorders, inflammation, autoimmune
diseases, ischemic conditions, immune-related
disorders, hyper-tension and neurological disorders.
During the course of present studies, ligand docking of
several Sulfamoylbenzamide derivatives into the
substrate-binding clefts of Plasmepsin-II from
Plasmodium falciparum and human cathepsin D has been
N
H
S NH2 O
O
N-(2,6-dimethylphenyl)-2-sulfamoylbenzamide
O
NH
S NH2 O
O
N-(pyridin-2-yl)-2-sulfamoylbenzamide
CD= human cathepsin D; PII= Plasmepsin II
cathepsin D inhibition (%)
80
70
60
50
40
30
20
10
0
N
Inhibition of CD
(IC50 in µM)
Docking in PII
(FlexX score)
-32.5±0.20 2.0±0.10 -27.9±0.10
Inhibtion of PII
(IC50 in µM)
Not active
-35.1±0.13 2.0±0.13 -29.5±0.10 Not active
-30.0±0.10 1.25±0.08 -26.1±0.15 Not active
-30.9±0.13 1.5±0.08 -24.0±0.10 Not active
-29.9±1.16 1.5±0.05 -26.9±0.10 Not active
cathepsin D inhibition by Sulfamoylbenzamide compounds
w4 w7 w24 w28 w30
0 0.5 1 1.5
Inhibitor concentration (Micromol)
Fig. 1: Human cathepsin D inhibition plots of Sulfamoylbenzamide compounds.
carried out. Enzyme inhibition assays were also
performed using top scoring compounds from virtual
screening. Interestingly, enzyme inhibition results showed
that Sulfa-moylbenzamide compounds are selective
inhibitors of human cathepsin D and not active against
Plasmepsin II.
Pak. J. Pharm. Sci., Vol.26, No.4, July 2013, pp.687-690
2

MATERIALS AND METHODS
In this study, virtual screening cum enzyme inhibition
assay was carried out to find the potent inhibitors of
Plasmepsin II and Cathepsin D.
Molecular docking
FlexX ligand docking software (version 2.0) ( Rarey et al.
1996) was utilized for docking using Pepstatin-A
complexed crystal coordinates of cathepsin D and
plasmepsin-II (Silva et al, 1996) (Baldwin et al. 1993;
Metcalf and Fusek 1993) (PDB id; 1M43 and 1LYB
respectively). FlexX ligand docking method is based on
incremental construction of ligand molecules from
smaller fragments in the cavity of a protein. The free
binding energy (∆G) of protein-ligand complex is utilized
to rank the generated docking results using a scoring
function similar to that developed by Bohm (Bohm 1994).
After completion of each docking experiment, top ten
docking solutions were saved and utilized for in-depth
analysis.
Enzyme inhibition assay
The enzyme activities of plasmepsin-II and cathepsin D
were measured as described earlier (Haque et al, 1999)
using fluorescence resonance energy transfer (FRET)
based assay with the fluorogenic substrate DABCYL-Glu-
Arg-Nle-Phe-Leu-Ser-Phe-Pro-EDANS (malaria FRET-1;
AnaSpec Inc., USA). Purified Plasmodium falciparum
plasmepsin-II was provided by Daniel E. Goldberg,
Howard Hughes Medical Institute, St. Louis, Missouri,
USA. Recombinant human liver cathepsin D was
purchased from Biodesign International, USA. The assay
was performed with plasmepsin-II/cathepsin D (1.2 nM)
and substrate (malaria FRET-1; 1.0 μM) in 0.1 M Sodium
acetate buffer pH 5.0, containing 0.01% Tween 20 and
10% Glycerol. Before the addition of substrate the DMSO
solution of Sulfamoylbenzamide derivatives were added
in the reaction mixture. The assays were performed with
5.0% final concentration of DMSO. The enzyme
inhibition experiments were performed (in triplicates) in
96 well plate format and readings were obtained on a
Perkin Elmer LS55 Fluorescence spectrometer with an
excitation and emission wavelengths of 336 and 490 nm
respectively. IC50 values were calculated by nonlinear
regression analysis from plots of percentage inhibition
versus inhibitor concentrations. The enzyme assays using
‘standard inhibitor’ Pepstatin-A (Sigma Inc, USA) was
performed in the same experimental manner as for
Sulfamoylbenzamide derivatives.
Waseem Ahmed et al
essentially as described by Francis and coworkers
(Francis et al, 1994). 200 microliter aliquots of late ring
stage cultures at 0.5% parasitemia were incubated with
various concentrations of either pepstatinA (Roche
Applied Sciences) or trans-epoxysuccinyl-L-leucylamino
(4-guanidine)-butane (E64) in hypoxanthine-free rich
medium for 42 h. 0.5 μCi of [3H]hypoxanthine (178.7
Ci/mmol; PerkinElmer) was added to the culture and the
incubation continued for 24 h. Cultures were harvested on
glass fiber paper, immersed in UltimaGold scintillation
counting mixture (PerkinElmer, USA) and counted in a
scintillation counter. The percentage of the inhibition of
[3H] hypoxanthine uptake was plotted against the drug
concentration and the curve was fitted using the modified
dose-response logistic equation in KaleidaGraph software.
RESULTS
IC50 values of five Sulfamoylbenzamide compounds
tested are in range of 1.25-2.0 μM. Cathepsin D inhibition
plot of Sulfamoylbenzamide compounds is demonstrated
in figure 1. IC50 value of N-(3-chlorophenyl)-2-sulfamoylbenzamide
is 1.25 μM, so it is identified as the most
potent of all tested Sulfamoylbenzamide compounds. In
case of Cathepsin D docking score was -29.9±1.16 to -
35.1±0.13 while from -24.0±0.10 to -29.5±0.10 in case of
Plasmepsin-II. Chemical structures, codes and inhibition
data of these compounds are given table 1. Anti
plasmodial activity of N-(3-chlorophenyl)-2-sulfamoylbenzamide
was also carried out to evaluate its
antiplasmodial behavior as the representative molecule of
the series. The IC50 was ≥3 µM which is insignificant.
DISCUSSION
Aspartic proteases play vital role in different cellular
processes. Regulation and inhibition of their activities are
of important consequences. Cathepsin D is overexpressed
in breast cancer and considered as a possible target for
anti-cancer drugs. Virtual screening of hundreds of
compounds pointed out Sulfamoylbenzamide derivatives
as potential Cathepsin D and/or Plasmepsin II inhibitors.
Subsequent enzyme inhibition assay of selected
Sulfamoylbenzamide derivatives revealed these
compounds as selective Cathepsin D inhibitors. It was
also noted that the docking score of theses compounds
was better in case of Cathepsin D as compared to
Plasmepsin-II. These findings supports the results of
plasmepsin II inhibition assay as sulfamoylbenzamide
derivatives are inactive against plasmepsin II.
Antiplasmodial assays
The antiplasmodial assays were carried out in the
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